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AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).
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Diabetes Mellitus , MicroARNs , Humanos , Represión Epigenética , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células Madre/metabolismo , ARN Interferente Pequeño/metabolismo , Cicatrización de Heridas/genética , Células Epiteliales/metabolismoRESUMEN
Air pollution poses a significant threat to human health, though a clear understanding of its mechanism remains elusive. In this study, we sought to better understand the effects of various sized particulate matter from polluted air on Alzheimer's disease (AD) development using an AD mouse model. We exposed transgenic Alzheimer's mice in their prodromic stage to different sized particulate matter (PM), with filtered clean air as control. After 3 or 6 months of exposure, mouse brains were harvested and analyzed. RNA-seq analysis showed that various PM have differential effects on the brain transcriptome, and these effects seemed to correlate with PM size. Many genes and pathways were affected after PM exposure. Among them, we found a strong activation in mRNA Nonsense Mediated Decay pathway, an inhibition in pathways related to transcription, neurogenesis and survival signaling as well as angiogenesis, and a dramatic downregulation of collagens. Although we did not detect any extracellular Aß plaques, immunostaining revealed that both intracellular Aß1-42 and phospho-Tau levels were increased in various PM exposure conditions compared to the clean air control. NanoString GeoMx analysis demonstrated a remarkable activation of immune responses in the PM exposed mouse brain. Surprisingly, our data also indicated a strong activation of various tumor suppressors including RB1, CDKN1A/p21 and CDKN2A/p16. Collectively, our data demonstrated that exposure to airborne PM caused a profound transcriptional dysregulation and accelerated Alzheimer's-related pathology.
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Human diabetic corneas develop delayed wound healing, epithelial stem cell dysfunction, recurrent erosions, and keratitis. Adenoviral gene therapy modulating c-Met, cathepsin F and MMP-10 normalized wound healing and epithelial stem cells in organ-cultured diabetic corneas but showed toxicity in stem cell-enriched cultured limbal epithelial cells (LECs). For a safer treatment, we engineered a novel nanobiopolymer (NBC) that carried antisense oligonucleotide (AON) RNA therapeutics suppressing cathepsin F or MMP-10, and miR-409-3p that inhibits c-Met. NBC was internalized by LECs through transferrin receptor (TfR)-mediated endocytosis, inhibited cathepsin F or MMP-10 and upregulated c-Met. Non-toxic NBC modulating c-Met and cathepsin F accelerated wound healing in diabetic LECs and organ-cultured corneas vs. control NBC. NBC treatment normalized levels of stem cell markers (keratins 15 and 17, ABCG2, and ΔNp63), and signaling mediators (p-EGFR, p-Akt and p-p38). Non-toxic nano RNA therapeutics thus present a safe alternative to viral gene therapy for normalizing diabetic corneal cells.
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Córnea/patología , Diabetes Mellitus/patología , Células Epiteliales/patología , Nanopartículas/química , Polímeros/química , ARN/uso terapéutico , Células Madre/patología , Cicatrización de Heridas , Adenoviridae/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Supervivencia Celular , Células Cultivadas , Córnea/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nanopartículas/ultraestructura , Oligonucleótidos Antisentido/farmacología , ARN/farmacología , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
HER2+ breast cancer is one of the most aggressive forms of breast cancer. The new polymalic acid-based mini nanodrug copolymers are synthesized and specifically characterized to inhibit growth of HER2+ breast cancer. These mini nanodrugs are highly effective and in the clinic may substitute for trastuzumab (the marketed therapeutic antibody) and antibody-targeted nanobioconjugates. Novel mini nanodrugs are designed to have slender shape and small size. HER2+ cells were recognized by the polymer-attached trastuzumab-mimetic 12-mer peptide. Synthesis of the nascent cell-transmembrane HER2/neu receptors by HER2+ cells was inhibited by antisense oligonucleotides that prevented cancer cell proliferation and significantly reduced tumor size by more than 15 times vs. untreated control or PBS-treated group. We emphasize that the shape and size of mini nanodrugs can enhance penetration of multiple bio-barriers to facilitate highly effective treatment. Replacement of trastuzumab by the mimetic peptide favors reduced production costs and technical efforts, and a negligible immune response.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Receptor ErbB-2 , Trastuzumab/farmacocinética , Anticuerpos Monoclonales Humanizados/administración & dosificación , Línea Celular Tumoral , Humanos , Nanopartículas/química , Péptidos/uso terapéutico , Trastuzumab/administración & dosificaciónRESUMEN
Multifunctional polymer nanoconjugates containing multiple components show great promise in cancer therapy, but in most cases complete analysis of each component is difficult. Polymalic acid (PMLA) based nanoconjugates have demonstrated successful brain and breast cancer treatment. They consist of multiple components including targeting antibodies, Morpholino antisense oligonucleotides (AONs), and endosome escape moieties. The component analysis of PMLA nanoconjugates is extremely difficult using conventional spectrometry and HPLC method. Taking advantage of the nature of polyester of PMLA, which can be cleaved by ammonium hydroxide, we describe a method to analyze the content of antibody and AON within nanoconjugates simultaneously using SEC-HPLC by selectively cleaving the PMLA backbone. The selected cleavage conditions only degrade PMLA without affecting the integrity and biological activity of the antibody. Although the amount of antibody could also be determined using the bicinchoninic acid (BCA) method, our selective cleavage method gives more reliable results and is more powerful. Our approach provides a new direction for the component analysis of polymer nanoconjugates and nanoparticles.
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Malatos/química , Nanoconjugados/química , Polímeros/química , Antineoplásicos/química , Sistemas de Liberación de Medicamentos , Cinética , Trastuzumab/químicaRESUMEN
Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semisynthetic materials or utilize potentially denaturing assembly conditions that are not optimal for sensitive biologics. Layer-by-layer (LbL) assembly of films uses benign conditions and can generate films with various release mechanisms including hydrolysis-facilitated degradation. These use components such as synthetic polycations that degrade into non-natural products. Herein we report the use of a naturally derived, biocompatible and degradable polyanion, poly(ß-l-malic acid), alone and in combination with chitosan in an LbL film, whose degradation products of malic acid and chitosan are both generally recognized as safe (GRAS) by the FDA. We have found that films based on this polyanion have shown sustained release of a model protein, lysozyme that can be timed from tens of minutes to multiple days through different film architectures. We also report the incorporation and release of a clinically used biologic, basic fibroblast growth factor (bFGF), which demonstrates the use of this strategy as a platform for controlled release of various biologics.
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Quitosano/química , Sistemas de Liberación de Medicamentos/métodos , Malatos/química , Muramidasa/química , Polímeros/química , Animales , Quitosano/metabolismo , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/metabolismo , Malatos/metabolismo , Ratones , Muramidasa/metabolismo , Células 3T3 NIH , Polímeros/metabolismoRESUMEN
OBJECT: The intraoperative clear delineation between brain tumor and normal tissue in real time is required to ensure near-complete resection without damaging the nearby eloquent brain. Tumor Paint BLZ-100, a tumor ligand chlorotoxin (CTX) conjugated to indocyanine green (ICG), has shown potential to be a targeted contrast agent. There are many infrared imaging systems in use, but they are not optimized to the low concentration and amount of ICG. The authors present a novel proof-of-concept near-infrared (NIR) imaging system using a standard charge-coupled device (CCD) camera for visualizing low levels of ICG attached to the tumors. This system is small, inexpensive, and sensitive. The imaging system uses a narrow-band laser at 785 nm and a notch filter in front of the sensor at the band. The camera is a 2-CCD camera, which uses identical CCDs for both visible and NIR light. METHODS: The NIR system is tested with serial dilution of BLZ-100 from 1 µM to 50 pM in 5% Intralipid solution while the excitation energy is varied from 5 to 40 mW/cm(2). The analog gain of the CCD was changed from 0, 6, and 12 dB to determine the signal-to-noise ratio. In addition to the Intralipid solution, BLZ-100 was injected 48 hours before euthanizing the mice that were implanted with the human glioma cell line. The brain was removed and imaged using the NIR imaging system. RESULTS: The authors' results show that the NIR imaging system using a standard CCD is able to visualize the ICG down to 50 nM of concentration with a high signal-to-noise ratio. The preliminary experiment on human glioma implanted in mouse brains demonstrated that BLZ-100 has a high affinity for glioma compared with normal brain tissue. Additionally, the results show that NIR excitation is able to penetrate deeply and has a potential to visualize metastatic lesions that are separate from the main tumor. CONCLUSIONS: The authors have seen that BLZ-100 has a very high affinity toward human gliomas. They also describe a small, cost-effective, and sensitive NIR system for visualizing brain tumors tagged using BLZ-100. The authors hope that the use of BLZ-100 along with NIR imaging will be useful to delineate the brain tumors in real time and assist surgeons in near-complete tumor removal to increase survival and reduce neurological deficits.
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Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/cirugía , Diagnóstico por Imagen/métodos , Verde de Indocianina , Venenos de Escorpión , Espectroscopía Infrarroja Corta/métodos , Animales , Diagnóstico por Imagen/instrumentación , Humanos , Verde de Indocianina/análogos & derivados , Ratones , Espectroscopía Infrarroja Corta/instrumentaciónRESUMEN
Multidrug regimens can sometimes treat recalcitrant diseases when single-drug therapies fail. Recapitulating complex multidrug administration from controlled release films for localized delivery remains challenging because their release kinetics are frequently intertwined, and an initial burst release of each drug is usually uncontrollable. Kinetic control over protein release is demonstrated by cross-linking layer-by-layer films during the assembly process. We used biodegradable and naturally derived components and relied on copper-free click chemistry for bioorthogonal covalent cross-links throughout the film that entrap but do not modify the embedded protein. We found that this strategy restricted the interdiffusion of protein while maintaining its activity. By depositing a barrier layer and a second protein-containing layer atop this construct, we generated well-defined sequential protein release with minimal overlap that follows their spatial distribution within the film.
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Proteínas/metabolismo , CinéticaRESUMEN
BACKGROUND: Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death. SCOPE OF REVIEW: In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses. MAJOR CONCLUSIONS: Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo. GENERAL SIGNIFICANCE: The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. This article is part of a Special Issue entitled Transferrins: molecular mechanisms of iron transport and disorders.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/administración & dosificación , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Receptores de Transferrina/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/metabolismo , Sistemas de Liberación de Medicamentos , Vectores Genéticos , Humanos , Hierro/metabolismoRESUMEN
Effective treatment of brain neurological disorders such as Alzheimer's disease, multiple sclerosis, or tumors should be possible with drug delivery through blood-brain barrier (BBB) or blood-brain tumor barrier (BTB) and targeting specific types of brain cells with drug release into the cell cytoplasm. A polymeric nanobioconjugate drug based on biodegradable, nontoxic, and nonimmunogenic polymalic acid as a universal delivery nanoplatform was used for design and synthesis of nanomedicine drug for i.v. treatment of brain tumors. The polymeric drug passes through the BTB and tumor cell membrane using tandem monoclonal antibodies targeting the BTB and tumor cells. The next step for polymeric drug action was inhibition of tumor angiogenesis by specifically blocking the synthesis of a tumor neovascular trimer protein, laminin-411, by attached antisense oligonucleotides (AONs). The AONs were released into the target cell cytoplasm via pH-activated trileucine, an endosomal escape moiety. Drug delivery to the brain tumor and the release mechanism were both studied for this nanobiopolymer. Introduction of a trileucine endosome escape unit resulted in significantly increased AON delivery to tumor cells, inhibition of laminin-411 synthesis in vitro and in vivo, specific accumulation in brain tumors, and suppression of intracranial glioma growth compared with pH-independent leucine ester. The availability of a systemically active polymeric drug delivery system that passes through the BTB, targets tumor cells, and inhibits glioma growth gives hope for a successful strategy of glioma treatment. This delivery system with drug release into the brain-specific cell type could be useful for treatment of various brain pathologies.
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Neoplasias Encefálicas/tratamiento farmacológico , Concentración de Iones de Hidrógeno , Malatos/uso terapéutico , Nanopartículas , Polímeros/uso terapéutico , Animales , Barrera Hematoencefálica , Neoplasias Encefálicas/patología , Endosomas/metabolismo , Infusiones Intravenosas , Malatos/administración & dosificación , Malatos/farmacocinética , Ratones , Ratones Desnudos , Polímeros/administración & dosificación , Polímeros/farmacocinéticaRESUMEN
Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aß) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.
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Enfermedad de Alzheimer , MicroARNs , Ácidos Nucleicos , Ratones , Animales , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Ácidos Nucleicos/uso terapéutico , Encéfalo/metabolismo , Barrera Hematoencefálica/metabolismo , Nanoconjugados/uso terapéutico , MicroARNs/uso terapéutico , Neuronas/metabolismo , Modelos Animales de Enfermedad , Ratones TransgénicosRESUMEN
Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(ß-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.
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Antibióticos Antineoplásicos , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina , Sistemas de Liberación de Medicamentos , Glioma/tratamiento farmacológico , Malatos , Nanoconjugados/química , Polímeros , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Femenino , Glioma/metabolismo , Glioma/patología , Humanos , Hidrazonas/química , Concentración de Iones de Hidrógeno , Malatos/química , Malatos/farmacocinética , Malatos/farmacología , Masculino , Polímeros/química , Polímeros/farmacocinética , Polímeros/farmacologíaRESUMEN
Polyethylene glycol or PEG has a long history of use in medicine. Many conventional formulations utilize PEG as either an active ingredient or an excipient. PEG found its use in biotechnology therapeutics as a tool to slow down drug clearance and shield protein therapeutics from undesirable immunogenicity. Nanotechnology field applies PEG to create stealth drug carriers with prolonged circulation time and decreased recognition and clearance by the mononuclear phagocyte system (MPS). Most nanomedicines approved for clinical use and experimental nanotherapeutics contain PEG. Among the most recent successful examples are two mRNA-based COVID-19 vaccines that are delivered by PEGylated lipid nanoparticles. The breadth of PEG use in a wide variety of over the counter (OTC) medications as well as in drug products and vaccines stimulated research which uncovered that PEG is not as immunologically inert as it was initially expected. Herein, we review the current understanding of PEG's immunological properties and discuss them in the context of synthesis, biodistribution, safety, efficacy, and characterization of PEGylated nanomedicines. We also review the current knowledge about immunological compatibility of other polymers that are being actively investigated as PEG alternatives.
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Portadores de Fármacos , Nanomedicina , Polietilenglicoles/química , Animales , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/inmunología , Sistemas de Liberación de Medicamentos , HumanosRESUMEN
After over a billion of vaccinations with messenger RNA-lipid nanoparticle (mRNA-LNP) based SARS-CoV-2 vaccines, anaphylaxis and other manifestations of hypersensitivity can be considered as very rare adverse events. Although current recommendations include avoiding a second dose in those with first-dose anaphylaxis, the underlying mechanisms are unknown; therefore, the risk of a future reaction cannot be predicted. Given how important new mRNA constructs will be to address the emergence of new viral variants and viruses, there is an urgent need for clinical approaches that would allow a safe repeated immunization of high-risk individuals and for reliable predictive tools of adverse reactions to mRNA vaccines. In many aspects, anaphylaxis symptoms experienced by the affected vaccine recipients resemble those of infusion reactions to nanomedicines. Here we share lessons learned over a decade of nanomedicine research and discuss the current knowledge about several factors that individually or collectively contribute to infusion reactions to nanomedicines. We aim to use this knowledge to inform the SARS-CoV-2 lipid-nanoparticle-based mRNA vaccine field.
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Anafilaxia , COVID-19 , Anafilaxia/etiología , COVID-19/prevención & control , Vacunas contra la COVID-19/efectos adversos , Humanos , Liposomas , Nanomedicina , Nanopartículas , ARN Mensajero/genética , SARS-CoV-2/genética , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
The ability to cross the blood-brain barrier (BBB) is critical for targeted therapy of the central nerve system (CNS). Six peptide vectors were covalently attached to a 50 kDa poly(ß-l-malic acid)-trileucine polymer forming P/LLL(40%)/vector conjugates. The vectors were Angiopep-2 (AP2), B6, Miniap-4 (M4), and d-configurated peptides D1, D3, and ACI-89, with specificity for transcytosis receptors low-density lipoprotein receptor-related protein-1 (LRP-1), transferrin receptor (TfR), bee venom-derived ion channel, and Aß/LRP-1 related transcytosis complex, respectively. The BBB-permeation efficacies were substantially increased ("boosted") in vector conjugates of P/LLL(40%). We have found that the copolymer group binds at the endothelial membrane and, by an allosterically membrane rearrangement, exposes the sites for vector-receptor complex formation. The specificity of vectors is indicated by competition experiments with nonconjugated vectors. P/LLL(40%) does not function as an inhibitor, suggesting that the copolymer binding site is eliminated after binding of the vector-nanoconjugate. The two-step mechanism, binding to endothelial membrane and allosteric exposure of transcytosis receptors, is supposed to be an integral feature of nanoconjugate-transcytosis pathways. In vivo brain delivery signatures of the nanoconjugates were recapitulated in mouse brains of normal, tumor (glioblastoma), and Alzheimer's disease (AD) models. BBB permeation of the tumor was most efficient, followed by normal and then AD-like brain. In tumor-bearing and normal brains, AP2 was the top performing vector; however, in AD models, D3 and D1 peptides were superior ones. The TfR vector B6 was equally efficient in normal and AD-model brains. Cross-permeation efficacies are manifested through modulated vector coligation and dosage escalation such as supra-linear dose dependence and crossover transcytosis activities.
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Enfermedad de Alzheimer , Barrera Hematoencefálica , Animales , Ratones , Barrera Hematoencefálica/metabolismo , Enfermedad de Alzheimer/patología , Nanoconjugados , Transcitosis , Péptidos/química , Polímeros/farmacología , Péptidos beta-Amiloides/metabolismoRESUMEN
Nanotechnology is the design and assembly of submicroscopic devices called nanoparticles, which are 1-100 nm in diameter. Nanomedicine is the application of nanotechnology for the diagnosis and treatment of human disease. Disease-specific receptors on the surface of cells provide useful targets for nanoparticles. Because nanoparticles can be engineered from components that (1) recognize disease at the cellular level, (2) are visible on imaging studies, and (3) deliver therapeutic compounds, nanotechnology is well suited for the diagnosis and treatment of a variety of diseases. Nanotechnology will enable earlier detection and treatment of diseases that are best treated in their initial stages, such as cancer. Advances in nanotechnology will also spur the discovery of new methods for delivery of therapeutic compounds, including genes and proteins, to diseased tissue. A myriad of nanostructured drugs with effective site-targeting can be developed by combining a diverse selection of targeting, diagnostic, and therapeutic components. Incorporating immune target specificity with nanostructures introduces a new type of treatment modality, nano-immunochemotherapy, for patients with cancer. In this review, we will discuss the development and potential applications of nanoscale platforms in medical diagnosis and treatment. To impact the care of patients with neurological diseases, advances in nanotechnology will require accelerated translation to the fields of brain mapping, CNS imaging, and nanoneurosurgery. Advances in nanoplatform, nano-imaging, and nano-drug delivery will drive the future development of nanomedicine, personalized medicine, and targeted therapy. We believe that the formation of a science, technology, medicine law-healthcare policy (STML) hub/center, which encourages collaboration among universities, medical centers, US government, industry, patient advocacy groups, charitable foundations, and philanthropists, could significantly facilitate such advancements and contribute to the translation of nanotechnology across medical disciplines.
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Antineoplásicos/uso terapéutico , Política de Salud/legislación & jurisprudencia , Política de Salud/tendencias , Nanomedicina/legislación & jurisprudencia , Nanomedicina/tendencias , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/tendencias , Humanos , Imagenología Tridimensional/métodos , Nanomedicina/métodos , Nanoestructuras/uso terapéutico , Medicina de Precisión/métodos , Medicina de Precisión/tendenciasRESUMEN
Research has increasingly focused on the delivery of high, often excessive amounts of drugs, neglecting negative aspects of the carrier's physical preconditions and biocompatibility. Among them, little attention has been paid to "small but beautiful" design of vehicle and multiple cargo to achieve effortless targeted delivery into deep tissue. The design of small biopolymers for deep tissue targeted delivery of multiple imaging agents and therapeutics (mini-nano carriers) emphasizes linear flexible polymer platforms with a hydrodynamic diameter of 4 nm to 10 nm, geometrically favoring dynamic juxtaposition of ligands to host receptors, and economic drug content. Platforms of biodegradable, non-toxic poly(ß-l-malic acid) of this size carrying multiple chemically bound, optionally nature-derived or synthetic affinity peptides and drugs for a variety of purposes are described in this review with specific examples. The size, shape, and multiple attachments to membrane sites accelerate vascular escape and fast blood clearance, as well as the increase in medical treatment and contrasts for tissue imaging. High affinity antibodies routinely considered for targeting, such as the brain through the blood-brain barrier (BBB), are replaced by moderate affinity binding peptides (vectors), which penetrate at high influxes not achievable by antibodies.
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Glioblastoma (GBM) is the most prevalent primary brain cancer in the pediatric and adult population. It is known as an untreatable tumor in urgent need of new therapeutic approaches. The objective of this work was to develop multifunctional nanomedicines to treat GBM in clinical practice using combination therapy for several targets. We developed multifunctional nanopolymers (MNPs) based on a naturally derived biopolymer, poly(ß-L-malic) acid, which are suitable for central nervous system (CNS) treatment. These MNPs contain several anticancer functional moieties with the capacity of crossing the blood-brain barrier (BBB), targeting GBM cells and suppressing two important molecular markers, tyrosine kinase transmembrane receptors EGFR/EGFRvIII and c-Myc nuclear transcription factor. The reproducible syntheses of MNPs where monoclonal antibodies are replaced with AP-2 peptide for effective BBB delivery were presented. The active anticancer inhibitors of mRNA/protein syntheses were Morpholino antisense oligonucleotides (AONs). Two ways of covalent AON-polymer attachments with and without disulfide bonds were explored. These MNPs bearing AONs to EGFR/EGFRvIII and c-Myc, as well as in a combination with the polymer-attached checkpoint inhibitor anti-PD-1 antibody, orchestrated a multi-pronged attack on intracranial mouse GBM to successfully block tumor growth and significantly increase survival of brain tumor-bearing animals.
RESUMEN
PURPOSE: Temozolomide (TMZ) is a pro-drug releasing a DNA alkylating agent that is the most effective drug to treat glial tumors when combined with radiation. TMZ is toxic, and therapeutic dosages are limited by severe side effects. Targeted delivery is thus needed to improve efficiency and reduce non-tumor tissue toxicity. METHODS: Multifunctional targetable nanoconjugates of TMZ hydrazide were synthesized using poly(ß-L-malic acid) platform, which contained a targeting monoclonal antibody to transferrin receptor (TfR), trileucine (LLL), for pH-dependent endosomal membrane disruption, and PEG for protection. RESULTS: The water-soluble TMZ nanoconjugates had hydrodynamic diameters in the range of 6.5 to 14.8 nm and ζ potentials in the range of -6.3 to -17.7 mV. Fifty percent degradation in human plasma was observed in 40 h at 37°C. TMZ conjugated with polymer had a half-life of 5-7 h, compared with 1.8 h for free TMZ. The strongest reduction of human brain and breast cancer cell viability was obtained by versions of TMZ nanoconjugates containing LLL and anti-TfR antibody. TMZ-resistant cancer cell lines were sensitive to TMZ nanoconjugate treatment. CONCLUSIONS: TMZ-polymer nanoconjugates entered the tumor cells by receptor-mediated endocytosis, effectively reduced cancer cell viability, and can potentially be used for targeted tumor treatment.
Asunto(s)
Antineoplásicos/administración & dosificación , Dacarbazina/análogos & derivados , Malatos/química , Nanopartículas , Polímeros/química , Línea Celular Tumoral , Dacarbazina/administración & dosificación , Humanos , Liposomas , Peso Molecular , TemozolomidaRESUMEN
Magnetic nanoparticles in general, and iron oxide nanoparticles in particular, have been studied extensively during the past 20 years for numerous biomedical applications. The main applications of these nanoparticles are in magnetic resonance imaging (MRI), magnetic targeting, gene and drug delivery, magnetic hyperthermia for tumor treatment, and manipulation of the immune system by macrophage polarization for cancer treatment. Recently, considerable attention has been paid to magnetic particle imaging (MPI) because of its better sensitivity compared to MRI. In recent years, MRI and MPI have been combined as a dual or multimodal imaging method to enhance the signal in the brain for the early detection and treatment of brain pathologies. Because magnetic and iron oxide nanoparticles are so diverse and can be used in multiple applications such as imaging or therapy, they have attractive features for brain delivery. However, the greatest limitations for the use of MRI/MPI for imaging and treatment are in brain delivery, with one of these limitations being the brain-blood barrier (BBB). This review addresses the current status, chemical compositions, advantages and disadvantages, toxicity and most importantly the future directions for the delivery of iron oxide based substances across the blood-brain barrier for targeting, imaging and therapy of primary and metastatic tumors of the brain.